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虫号: 862014
注册: 2009-10-02

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The oxidation was carried out in 23 mL of a phosphate
buffer (100 mm) containing 2 mm EDTA, 3 mm Cys,
0.15 mm Cys2 and 6 m GunHCl with a peptide concentration
of 100 mg/mL. The reaction mixture was dialysed
(2 h each) against 1 L of the same buffer containing 4 m
GunHCl (2 h), 2 m GunHCl (2 h) and the buffer without
GunHCl (20 h). The fully oxidized product was then purified
by HPLC (Vydac C18, 103250 mm, 300 A° , 5 mm, flow rate
3 mL/min; eluent A, 0.07% TFA; eluent B, 0.07% TFA in
MeCN–H2O 80 : 20; gradient: 10–70% B in 60 min, UV
detection at 215 nm). Purity was checked on a Nucleosil
C18 PPN column as described above and by capillary
electrophoresis (P/ACE System 2000, Beckman, Munich,
Germany; fused silica capillary, 47 cm375 mm, uncoated;
0.1 m phosphate buffer containing 0.02% hydroxymethylpropylcellulose,
pH 2.5; constant current 120 mA; UV detection
at 200 nm). The molecular weight determined for
LEAP-1/hepcidin by ESIMS was 2789.5 (Mr calc. 2789.3).
Trypsin cleavage of oxidized b-defensins
A sample of 300 mg of the purified defensins was dissolved
in 500 mL of 100 mm Tris-HCl. A solution of 12.5 mg trypsin
in 1 mm HCl (25 mL) was added. After 24 h at 368C, the
reaction was stopped by separation of the mixture (100 mL)
on an analytical Vydac C18 HPLC column. The tryptic
fragments detected were collected and analysed by ESIMS,
and the amino acid sequences of selected defensin fragments
were determined by Edman degradation (494 Procise peptide
sequencer, PE Biosystems, Weiterstadt, Germany).

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